Methods for labeling biomolecules with fluorescent dyes and affinity tags have become indispensable tools in the modern life sciences, and for those conjugation strategies it is important that they allow the site-specific post-synthetic coupling of complex molecules under mild conditions.
While for many years NHS ester chemistry dominated both protein and nucleic acid functionalization (G. T. Hermanson, Bioconjugate Techniques, Academic Press. San Diego, 1996), cycloaddition reactions have gained considerable importance. One of the most versatile post-synthetic labeling methods for DNA oligonucleotides is the copper(I)-catalyzed azide alkyne “click” reaction developed by Sharpless in 2001 (H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056-2075; Angew. Chem. Int. Ed. 2001, 40, 2004-2021). However, copper will be noxious for living cells above certain concentrations.
The first study on nucleic acid functionalization by Diels-Alder reaction used anthracene-modified oligonucleotides and maleimide dienophiles. While the reaction proceeded with high selectivity under mild conditions, the low reactivity required huge (>500-fold) excess of dienophile for appreciable product formation (B. Seelig, A. Jäschke, Tetrahedron Lett. 1997, 38, 7729-7732; b) B. Seelig, A. Jäschke, Chem. Biol. 1999, 6, 167-176). Consequently, only very few laboratories developed or applied normal electron demand Diels-Alder bioconjugations.
Since the aforementioned technologies have several disadvantages for labelling nucleic acid molecules, there is a further need for a method of post-synthetic modification of nucleic acids, preferably DNA, which is easy, cheap and works reliable also when larger nucleic acid molecules have to be labelled. Such a method should be suitable for small chemically synthesized oligonucleotides as well as for longer enzymatically amplified DNA strands.